Gene of porcine beta casein, a promoter of the same and the use thereof

ABSTRACT

The present invention provides a porcine beta-casein gene, a porcine beta-casein gene promoter, an expression vector comprising the same promoter, and a method for the production of a target protein using the same expression vector. The promoter of the present invention facilitates mammary gland-specific expression of the target protein and therefore can be useful for high-concentration production of beneficial proteins in milk.

RELATED APPLICATIONS

This application is the National Stage of International Application. No.PCT/KR2008/007823, filed 31 Dec. 2008, which claims benefit of priorityto KR 10-2008-0062767, filed 30 Jun. 2008, entitled “A GENE OF PORCINEBETA CASEIN, A PROMOTER OF THE SAME AND THE USES THEREOF.”

TECHNICAL FIELD

The present invention relates to a porcine beta-casein gene, a porcinebeta-casein gene promoter, an expression vector comprising the same, anda method for the production of a target protein using the same.

BACKGROUND ART

As an attempt to achieve maximum production of beneficial proteins (suchas EPO with high economic value-added) in the medicinal field, massproduction methods using cell culture techniques have been mainly used.

Korean Patent Application No. 94-12082 discloses an expression vectorcontaining a modified recombinant human erythropoietin (rhEPO) gene.Despite feasibility of mass production of EPO in the animal cell lineCOS-7 (ATCC CRL 1651, African Green Monkey Kidney Cell) transformed withthe same expression vector, this technique disadvantageously suffersfrom a cumbersome need of continuous transformation, which makes itunsuitable for industrial-scale production of a target protein. Further,Korean Patent No. 10-0232640 and Korean Patent Application PublicationNo. 1999-0075254 also disclose the production of EPO by transgenic cellline culture. However, these cell culture methods still suffer fromdisadvantages such as high production costs due to use of animal bloodas a culture medium, and requirement of expert and sophisticatedknowledge in the culture technique.

On the other hand, the production of beneficial proteins usingtransgenic animals is attracting a great deal of interest due to havingadvantages such as easy and convenient production, isolation andpurification of target proteins and maintenance of superior activity, ascompared to conventional cell culture techniques, because the targetproteins are contained in body fluids secreted by animals. For example,Korean Patent Application Publication No. 2004-0081456 discloses atransgenic animal for the production of EPO in porcine milk, using awhey acidic milk protein promoter (WAP).

As a result of a variety of extensive and intensive studies andexperiments to solve the problems as described above and to develop amammary gland-specific promoter with high-efficiency expression of atarget protein in milk, the inventors of the present invention succeededin sequencing of a beta-casein gene and a promoter thereof. The presentinvention has been completed based on this finding.

DISCLOSURE OF THE INVENTION

Technical Problem

The present invention is intended to provide a porcine beta-casein geneand a promoter thereof, and a method for mass production of a targetprotein using the same.

Technical Solution

The present invention provides a porcine beta-casein gene.

The beta-casein gene of the present invention specifically comprises asequence as set forth in SEQ ID NO: 1, and the sequence of SEQ ID NO: 1contains a promoter, and a sequence of a 3′ untranslated region (UTR).

Further, the present invention provides a promoter of SEQ ID NO: 2corresponding to a sequence of 1 to 5480 contiguous nucleotides, amongthe sequence of SEQ ID NO: 1, and the promoter is situated at the 5′side of a structural gene to thereby control expression of thestructural gene.

The porcine beta-casein gene or promoter of the present invention may beone selected from functional equivalents thereof having one or more ofdisruption, deletion, insertion, point, substitution, nonsense,missense, polymorphism and rearrangement mutations in the sequence ofSEQ ID NO: 1 or SEQ ID NO: 2.

Further, the present invention provides an expression vector comprisingan entire or partial promoter sequence of SEQ ID NO: 2. Preferably, theexpression vector of the present invention contains a sequence of SEQ IDNO: 3 or SEQ ID NO: 4. The sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4 serves as a promoter through the incorporation thereof into thevector and is referred to herein as a promoter sequence or porcinebeta-casein gene promoter sequence. As used herein, the term “porcinebeta-casein gene promoter” refers to a promoter derived from a porcinebeta-casein gene.

SEQ ID NO: 3 and SEQ ID NO: 4 respectively correspond to a sequenceconsisting of 67-5299 nucleotides and a sequence consisting of 561-5480nucleotides, among an entire genomic sequence of a porcine beta-caseingene of SEQ ID NO: 1, and contains in common a sequence consisting of561-5299 nucleotides among the sequence of SEQ ID NO: 1 and an exon 1region.

If necessary, the expression vector of the present invention mayadditionally contain regulatory factors at suitable sites or locithereof. Examples of the regulatory factors may include anotherpromoter, enhancer, selective marker, 5′-untranslated region (UTR),3′-UTR, polyadenylation signal, ribosome-binding sequence, sequence(s)capable of being inserted into a specific site of a genome, intron andwoodchuck hepatitis virus posttranscriptional regulatory element (WPRE).Incorporation of such additional elements into the expression vectorwill provide various advantages such as easy and convenient constructionof a transgenic cell line of interest, and maximized and stableexpression of target proteins.

The selective marker is preferably a neomycin-resistant gene or thelike. The selective marker may be one excised from a commerciallyavailable vector. The neomycin-resistant gene is a gene conferringresistance to G418 which is a reagent used in the construction of a cellline, and it may serve as an efficient selective marker upon theconstruction of an animal cell line that expresses a target proteinunder the control of a promoter.

The insulator is a factor that assists in the action of a regulatoryfactor adjacent to the promoter and facilitates position-independentexpression of a protein. The insulator factor allows for stableexpression of the protein under the control of a promoter. The insulatormay be one excised from a commercially available vector.

WPRE is a regulatory factor that can contribute to the stabilization ofmRNA molecules to thereby augment synthesis of proteins. This regulatorenables high expression of proteins under the control of a promoter.WPRE may also be a truncated one derived from a commercially availablevector.

The expression vector of the present invention may further comprise asequence as set forth in SEQ ID NO: 5. The sequence of SEQ ID NO: 5forms a 3′ arm of the vector and assists in easy construction of atransgenic cell line, and maximization and stabilization of targetprotein expression.

SEQ ID NO: 5 corresponds to a sequence ranging from nucleotide 10474 tonucleotide 15485, among an entire genomic sequence of the porcinebeta-casein gene of SEQ ID NO: 1 and it contains an exon 9 region.

Positions of sequences of SEQ ID NOs: 3, 4 and 5 among an entire genomicsequence of the porcine beta-casein gene are as shown in FIG. 1.

The vector of the present invention is preferably constructed to containthe sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 5.

Specifically, the vector of the present invention has a cleavage map asshown in FIG. 2. The pBC 1-Pig β casein vector was deposited with theKorean Collection for Type Cultures (KCTC), the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Daejon, Korea), underAccession Number KCTC 11327BP. The expression vector pBC1-Pig β caseinof the present invention has a pBC1 vector as a basic backbone, to whicha neomycin-resistant gene was fused as a selective marker.

The expression vector of the present invention may express a targetprotein by further incorporation of a target protein-encoding sequenceat a 3′ side of the promoter sequence.

The target protein is an industrially applicable beneficial protein andmay be any protein that is used, for example, as an active ingredient ofpharmaceuticals. Examples of the target protein may include EPO(erythropoietin), aldosterone, adrenocorticotropin, blood clottingfactors, gonadotropin, insulin, prolactin, and vasopressin. Preferred ishEPO.

The present invention provides a vector having a cleavage map of FIG. 3,as a preferable example of an expression vector harboring aneomycin-resistant gene, an insulator, WPRE, and the like. Specifically,the pBC1-Pig β casein+hEPO-WPRE vector was deposited with the KoreanCollection for Type Cultures (KCTC), the Korean Research Institute ofBioscience and Biotechnology (KRIBB, Daejon, Korea), under AccessionNumber KCTC 11328BP.

The expression vector pBC1-Pig β casein+hEPO-WPRE has a pBC1 vector as abasic backbone, wherein an hEPO-encoding gene is fused to a 3′ side ofthe promoter region of the present invention, and WPRE is fused to a 3′side of the hEPO gene

The expression vector of the present invention may be constructed in theform of a knock-in vector.

In the context of the present invention, the knock-in vector is a vectorcapable of inserting a target gene into a specific site or locus of agenome, and it contains a sequence homologous to a particular gene to betargeted, so as to result in homologous recombination therebetween. Theknock-in vector of the present invention is a beta-casein targetingvector where a target protein-encoding nucleic acid sequence is insertedinto a beta-casein gene present on the genome.

The knock-in vector of the present invention is preferably constructedto contain a sequence of SEQ ID NO: 4 and a sequence of SEQ ID NO: 5.

The knock-in vector may be constructed to select transgenic cells usinga positive and/or negative selective marker, if necessary. The selectivemarker is intended to select vector-transformed cells and may employgenes capable of conferring selectable phenotypes, such as drugresistance, nutritional auxotrophy, resistance to cytotoxic agents, andexpression of surface proteins. The selective marker may be broadlyclassified into a positive selective marker and a negative selectivemarker.

As used herein, the term “positive selective marker” refers to a genethat makes cells expressing the positive selective marker to surviveagainst a selective agent, so that it is capable of conferring positiveselective characteristics for the cells expressing that marker. Examplesof the positive selective marker may include neomycin (Neo)-resistantgene, hygromycin (Hyg)-resistant gene, etc.

The term “negative selective marker” refers to a gene which removescells with random-integration, so that it is capable of conferringnegative selection characteristics for the cells expressing that marker.Examples of the negative selective marker may include Herpes simplexvirus-thymidine kinase (HSV-tk) gene, hypoxanthine phosphoribosyltransferase (Hprt) gene, cytosine deaminase gene, Diphtheria toxin gene,etc. The negative selective marker is positioned at the 5′ terminus ofthe promoter region or at the 3′ terminus of the 3′ arm.

The positive selective marker and the negative selective marker may haveindependent promoters, poly(A), and the like. Examples of the promoterthat can be used in the present invention may include simian virus 40(SV40), mouse mammary tumor virus (MMTV) promoter, HIV long terminalrepeat (LTR) promoter, Moloney virus, Cytomegalovirus (CMV) promoter,Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter,phosphoglycerate kinase (PGK) promoter, etc.

When homologous recombination takes place between the knock-in vector ofthe present invention and the beta-casein gene on the genome, a targetprotein-encoding nucleic acid on the vector is integrated into thebeta-casein genomic gene of the host cell and is then expressed insteadof the beta-casein protein of the host cell.

The present invention provides a vector having a cleavage map of FIG. 4,as a preferable example of a knock-in vector employing aneomycin-resistant gene as a positive selective marker and Herpessimplex virus-thymidine kinase (HSV-tk) as a negative selective marker.

Specifically, the Pig β casein−hEPO knock-in vector was deposited withthe Korean Collection for Type Cultures (KCTC), the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Daejon, Korea), underAccession Number KCTC 11329BP.

The Pig β casein−hEPO knock-in vector has a Lox A vector as a basicbackbone, wherein hEPO is fused to a 3′ side of the promoter (referringto the Pig β casein 5′ arm region of FIG. 4), a neomycin-resistant geneas a positive selective marker is fused to a 3′ side of hEPO, a 3′ arm(referring to the Pig β casein 3′ arm of FIG. 4) is fused to a 3′ sideof the neomycin-resistant gene, and a Herpes simplex virus-thymidinekinase (HSV-tk) gene is fused to a 3′ side of the 3′ arm.

The vector of the present invention may be constructed by anyconventional gene recombination technique well-known in the art.Site-specific DNA cleavage and splicing may be carried out usingconventional enzymes known in the art.

Further, the present invention provides an animal somatic celltransformed by introduction of the expression vector of the presentinvention.

The animal somatic cell to which the vector of the present inventionwill be introduced may be a primary, secondary or permanent cell derivedfrom suitable animals including pigs.

Intracellular introduction of the vector of the present invention may becarried out by any conventional intracellular introduction method ofnucleic acids, that is, techniques known in the art, such aselectroporation, calcium phosphate co-precipitation, retroviralinfection, microinjection, DEAE-dextran facilitated transfection,cationic liposome-mediated transfection, etc. When it is desired toperform intracellular introduction of a vector, the vector may beintroduced in the form of a linearized vector or in the form of aplasmid-free linearized vector, by digestion of a circular vector withsuitable restriction enzymes.

The promoter gene of the present invention specifically expresses atarget protein only in mammary gland tissues. Casein accounts for 90% ofprotein components in porcine milk and is broadly categorized intoalpha-, beta- and gamma-casein. Since beta-casein contributes to aconsiderable portion of protein components, amounting to 27%, the vectoremploying the porcine beta-casein promoter may be constructed to exhibitmammary gland-specific expression of exogenous target proteins inlactating animals, particularly pigs.

Further, the present invention provides an animal embryo constructed bynuclear transfer of a nucleus of an animal somatic cell transformed withthe expression vector of the present invention into an enucleated egg.

As used herein, the term “nuclear transfer” refers to implantation of acell nucleus into an enucleated egg. The offspring produced byimplantation of the nuclear-transferred fertilized egg (or embryo) aregenetically completely identical clones because genetic materials of anuclear donor cell were thoroughly and intactly transferred into anuclear recipient cytoplasm.

Further, the present invention provides a transgenic animal obtained byimplantation of an animal embryo of the present invention.

Examples of the animals that can be transformed with the expressionvector of the present invention may include all kinds of lactatinganimals including pig, mouse, cow, sheep, and goat.

Production of a transgenic animal using the expression vector of thepresent invention is carried out by a conventional method known in theart.

For example, when an animal to be transformed is a mouse, embryos (orfertilized eggs) are collected from a healthy individual, and theexpression vector of the present invention is introduced into theembryos. Thereafter, a pseudopregnant mouse is obtained using avasoligated mouse, the embryos are implanted into an oviduct of thepseudopregnant mouse as a surrogate mother (or recipient), andtransgenic mice are then selected from among the offspring obtained fromthe surrogate mother.

When an animal to be transformed is a pig, porcine follicular oocytesare collected from a healthy animal and cultured in an in vitromaturation (IVM) medium. Further, the expression vector of the presentinvention is introduced into donor somatic cells collected and culturedfrom the porcine fetus, and somatic cells with integration of the vectorare selected and cultured. The in vitro matured eggs are enucleated, thedonor cells are injected into the enucleated space of the egg cells fromwhich nuclei were removed, and the donor cells and the cytoplasm of thenuclear-transferred eggs are fused by an electrofusion technique,followed by in vitro culture of the fusion. The resulting cloned embryosare implanted into the recipient pigs which were subjected tosuperovulation treatment, and the transgenic pigs are then selected fromamong the offspring obtained from the recipient pigs.

Thereafter, milk is collected from the individual where correcttransformation was confirmed, and a target protein is isolated andpurified therefrom to produce a final protein (A. Gokana, J. J.Winchenn, A. Ben-Ghanem, A. Ahaded, J. P. Cartron, P. Lambin (1997)Chromatographic separation of recombinant human erythropoietin isoforms,Journal of Chromatography, 791, 109-118).

In the production of the target protein of the present invention,isolation and purification of the protein may be carried out by aconventional method known in the art, for example filtration orchromatography.

The thus-constructed transgenic animal of the present invention canexpress the target protein in milk.

Therefore, the porcine beta-casein gene of the present invention, thepromoter thereof, and the expression vector and transgenic animal usingthe same can be beneficially used for the production of target proteins.

Details relating to genetic engineering techniques in the presentinvention can be found in the following literature: Sambrook, et al.Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001); and Frederick M. Ausubel et al.,Current Protocols in Molecular Biology volume 1, 2, 3, John Wiley &Sons, Inc. (1994).

Advantageous Effects

A porcine beta-casein gene promoter facilitates mammary gland-specificexpression of a target protein. Therefore, a promoter of the presentinvention and an animal transformed with an expression vectorconstructed using the same promoter enable high-concentration secretionof the target protein in milk, which consequently will provide benefitsfor the production of useful proteins that are medically andpharmaceutically valuable.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows positions of sequences found by PCR amplification duringsequencing of a porcine beta-casein in accordance with the presentinvention, as depicted in an entire sequence of porcine beta-casein.

FIG. 2 shows a structure of a pBC1-Pig β casein expression vector inaccordance with the present invention.

FIG. 3 shows a structure of a pBC1-Pig β casein+hEPO-WPRE expressionvector in accordance with the present invention.

FIG. 4 shows a structure of a Pig β casein−hEPO knock-in vector inaccordance with the present invention.

MODE FOR INVENTION

Now, the present invention will be described in more detail withreference to the following Examples. These examples are provided onlyfor illustrating the present invention and should not be construed aslimiting the scope and spirit of the present invention.

Example 1 Isolation and Cloning of Porcine Beta-Casein Gene

In order to construct a mammary gland-specific vector of the presentinvention, a porcine beta-casein gene was sequenced using bacterialartificial chromosome (BAC) clones provided by The National LivestockResearch Institute (Korea).

1) Sequencing of Porcine Beta-Casein Using BAC Clones

For sequencing of a porcine beta-casein gene, a primer pair consistingof 5′-TCTTGAAAACCTACCAAGTGC-3′ (forward, SEQ ID NO: 6) and5′-ATTCGTACAACACGGTCATTT-3′ (reverse, SEQ ID NO: 7) was constructed withreference to a sequence of a porcine beta-casein promoter region (5.5kb) available from The National Center for Biotechnology Information(NCBI) (http://www.ncbi.nlm.nih.gov, AY452035). The sequence of SEQ IDNO: 6 corresponds to a sequence of 2719 to 2739 nucleotides among thesequence of SEQ ID NO: 1, and the sequence of SEQ ID NO: 7 correspondsto a sequence of 3284 to 3304 nucleotides among the sequence of SEQ IDNO: 1. Using this primer set, four clones (155F1, 188A9, 616B6, and874E5) were obtained by PCR amplification from The National LivestockResearch Institute (Korea). PCR was carried out as follows: one cycle ofdenaturation at 94° C. for 5 minutes; and 35 cycles of denaturation at94° C. for 30 seconds, primer annealing at 56° C. for 30 seconds andelongation at 72° C. for 30 seconds.

In order to screen a porcine beta-casein gene using four clones thusobtained, primer sequences (SEQ ID NO: 8 to SEQ ID NO: 13) for use inPCR amplification of the porcine beta-casein were constructed withreference to portions that are highly homologous and well conservedamong different species, obtained by comparing beta-casein cDNAsequences of human, cow, horse and mouse whose beta-casein sequenceswere already known.

TABLE 1 SEQ ID Names Primers NO ATG up 5′-AGAGAACTCTATCCAATCACTT-3′ 8forward ATG up 5′-GCAAGGATGAGGAGCTTCAT-3′ 9 reverse ATG down5′-ATGAAGCTCCTCATCCTTGC-3′ 10 forward ATG down5′-TCTGCTGGAGATTTAGGGAAG-3′ 11 reverse 3′ down5′-CTTCCCTAAATCTCCAGCAGA-3′ 12 forward 3′ down5′-GTTGTCACATTTCCAGTCACA-3′ 13 reverse

PCR amplification was carried out using the constructed primers and616B6 out of four BAC clones as a template. The resulting PCR productsof 0.5 kb (SEQ ID NO: 14), 4.0 kb (SEQ ID NO: 15) and 1.7 kb (SEQ ID NO:16) were each cloned into a pGEM-T vector, followed by continuoussequencing.

TABLE 2 SEQ ID Primers for sequencing NO For 4.0 kb5′-CCTGTGTCTATTGAACAGAGA-3′ 17 5′-AGAAGGAAGAACTCAATGCAT-3′ 185′-AATGGTACATCACTAAACTTTG-3′ 19 5′-GGTGTGATCTGTTTTCTAGGA-3′ 205′-GTGTGACAACTTGCATAGTTAT-3′ 21 For 1.7 kb 5′-GTCCAAGTTTATTCACTGTGC-3′22

Positional structures of PCR-screened sequences are as shown in FIG. 1.

After specific primers were constructed using the analyzed sequences, a6.5-kb fragment (SEQ ID NO: 31) at a 3′ side of the porcine beta-caseinwas sequenced by repetitive sequencing (SEQ ID NO: 23 to SEQ ID NO: 30)using the BAC clone 616B6 as a template.

TABLE 3 SEQ ID NO Primers for sequencing 23 5′-TGGTGCTGTATAAGTTAGGCT-3′24 5′-TAAGTCCTTGACATTGCTGAG-3′ 25 5′-CTTTGCATCGTCTCTTCTGG-3′ 265′-ACCCAATACTCCTAACAATGC-3′ 27 5′-CCTCAGAAACTGTAATAGTTG-3′ 285′-CCTTTCTGCTGTATCCTCAC-3′ 29 5′-CAGGATGTCGCTTGAACAAG-3′ 305′-GGAGACTAGTGTCACCAAAC-3′

2) Sequencing of Beta-Casein from Berkshire Pigs

Based on a DNA sequence of the porcine beta-casein obtained from Bacclones, a sequence of beta-casein was sequenced from a genomic DNA ofBerkshire pigs. The searched beta-casein 12.6-kb fragment and thealready-sequenced 5.5-kb fragment were ligated and the resulting 17.7-kbsequence fragment was divided into five parts (3.6 kb, 3.9 kb, 4.2 kb,3.3 kb, 3.8 kb) which correspond to primer sequences (SEQ ID NO: 32 toSEQ ID NO: 41) for use in PCR amplification (PT-200, BIO-RAD). PCR wascarried out as follows: one cycle of denaturation at 94° C. for 5minutes; and 35 cycles of denaturation at 94° C. for 30 seconds, primerannealing at 56° C. for 30 seconds and elongation at 72° C. for 4minutes.

The resulting PCR products were each cloned into a pGEM-T vector,followed by sequencing. Analysis of sequences was conducted by Sogent(Korea) using a Bioedit program.

TABLE 4 SEQ ID Primers NO Forward 5′-ATCAGATGTTATTTTATGTGGCTAATC-3′ 323.6 kb Reverse 5′-ATTTTTAGAAGAAGAGCATATTTGTCA-3′ 33 3.6 kb Forward5′-AGGGTATTTGTGGGTATTTAAGATAGT-3′ 34 3.9 kb Reverse5′-AATGGTACATCACTAAACTTTGACTCT-3′ 35 3.9 kb Forward 5′-TCTCTCTCTATATTAACCTCATTCACT 36 4.2 kb G-3′ Reverse5′-CCTTTTGTGATCATGATATAGTAAACA-3′ 37 4.2 kb Forward5′-CAGTTGCCTATACACTTACACTTGAT-3′ 38 3.3 kb Reverse5′-AGTCATGGTCTAAAGTGGAATGGGA-3′ 39 3.3 kb Forward5′-AACTAACATTTCTTCTCTTAGGTATAC-3′ 40 3.8 kb Reverse5′-AAAGGATTATATGCTATCTAATATAGAG 41 3.8 kb T-3′

As a result, the porcine beta-casein genomic DNA sequence (SEQ ID NO: 1)of the Berkshire pig and sequence information thereof were successfullyacquired.

The sequence of SEQ ID NO: 1 is an entire genomic sequence of theporcine beta-casein gene and has a length of 17660 bp. In the sequenceof SEQ ID NO: 1, the structural gene region is a sequence ranging fromnucleotide 3067 to nucleotide 11460, the initiation codon is a sequenceranging from nucleotide 5501 to nucleotide 5503, and the terminationcodon is a sequence ranging from nucleotide 10381 to nucleotide 10383.In addition, the 5′ UTR region is a sequence ranging from nucleotide3067 to nucleotide 3087 and from nucleotide 5489 to nucleotide 5500, the3′ UTR region is a sequence ranging from nucleotide 10384 to nucleotide10419 and from nucleotide 11154 to nucleotide 11460, and the poly(A)signal region is a sequence ranging from nucleotide 11440 to nucleotide11445. The exon region is a sequence ranging from nucleotide 3074 tonucleotide 3087, from nucleotide 5489 to nucleotide 5551, fromnucleotide 6287 to nucleotide 6313, from nucleotide 6432 to nucleotide6458, from nucleotide 7784 to nucleotide 7807, from nucleotide 7902 tonucleotide 7946, from nucleotide 9253 to nucleotide 9771, fromnucleotide 10378 to nucleotide 10419, and from nucleotide 11154 tonucleotide 11460. The intron region is a sequence ranging fromnucleotide 3088 to nucleotide 5488, from nucleotide 5552 to nucleotide6286, from nucleotide 6314 to nucleotide 6431, from nucleotide 6459 tonucleotide 7783, from nucleotide 7808 to nucleotide 7901, fromnucleotide 7947 to nucleotide 9252, from nucleotide 9772 to nucleotide10377, and from nucleotide 10420 to nucleotide 11153. The CDS (codingsequence) is a sequence ranging from nucleotide 5501 to nucleotide 5551,from nucleotide 6287 to nucleotide 6313, from nucleotide 6432 tonucleotide 6458, from nucleotide 7784 to nucleotide 7807, fromnucleotide 7902 to nucleotide 7946, from nucleotide 9253 to nucleotide9771, and from nucleotide 10378 to nucleotide 10383.

In addition, a beta-casein amino acid sequence (SEQ ID NO: 42) wasanalyzed.

The analyzed porcine beta-casein sequence and information thereof wereregistered in NCBI (EU025876).

Example 2 Construction of pBC1-Pig β Casein Cloning Vector

A cloning vector was constructed by respectively replacing a goatbeta-casein promoter region and a 3′ genomic DNA region with thepromoter sequence and the 3′ arm sequence in a vector havingsubstitution of an ampicillin-resistant gene of a pBC1 vector(Invitrogen, USA) with a neomycin-resistant gene {A “neo” gene capableof conferring drug resistance to G418 was obtained from a pEGFP-N1vector (Clontech, USA) by amplification of a 1.9-kb PCR product (SEQ IDNO: 45) using a forward primer 5′-GCGGCCGCGCGCGTCAGGTGGCAC-3′ (SEQ IDNO: 43) and a reverse primer 5′-CGATCGGACGCTCAGTGGAACGAAAACTC-3′ (SEQ IDNO: 44), and was then cloned into a pGEM T-easy vector. The 1.9-kb neogene cloned into the T-vector was digested with restrictionendonucleases NotI and PvuI to prepare an insert. In addition, an ampgene (ampicillin-resistance gene) region of the pBC1 vector was removedby NotI and PvuI cleavage to prepare a vector. The resulting insertfragment and vector part were ligated to construct a pBC1 vector intowhich the neo gene (neomycin-resistance gene) was inserted}.

The promoter sequence 5.3 kb (SEQ ID NO: 3) and the 3′ arm sequence 5.0kb (SEQ ID NO: 5) were subjected to PCR amplification (PT-200, BIO-RAD)using primer sequences (SEQ ID NO: 46 to SEQ ID NO: 49). PCR was carriedout as follows: one cycle of denaturation at 94° C. for 5 minutes; and35 cycles of denaturation at 94° C. for 30 seconds, primer annealing at56° C. for 30 seconds and elongation at 72° C. for 5 minutes. Each ofthe resulting PCR products was cloned into a pGEM-T vector (Promega,USA).

TABLE 5 SEQ ID Primers NO Forward primer 5′-GGATCCGCTATGCAATCTCATG 46for promoter GAAAG-3′ amplification Reverse primer5′-CTCGAGTGACCAGGGTCAACAT 47 for promoter CTACT-3′ amplificationForward primer 5′-CTCGAGCTGCACTTCATTCTCC 48 for 3′ arm TGGATAA-3′amplification Reverse primer 5′-GCGGCCGCTTACAGTAAGACCT 49 for 3′ armTCAGGAGCA-3′ amplification

In order to avoid possible BamHI digestion, two BamHI sites (GGATCC)present in the porcine beta-casein promoter sequence were subjected torepetitive point mutations as follows. For introduction of pointmutations, one of two restriction sites was first selected and thecorresponding primer was constructed. The pGEM-T vector DNA containing aporcine beta-casein 5′ promoter region was purified and then subjectedto PCR amplification using 20 ng of template DNA and a pair of pointmutation primers. PCR was carried out as follows: one cycle ofdenaturation at 95° C. for 30 seconds; and 15 cycles of denaturation at95° C. for 30 seconds, primer annealing at 55° C. for 1 minute andelongation at 72° C. for 8.5 minutes. In order to eliminate the template(with no introduction of point mutation) DNA, 1 μl of Mutazyme™ wasadded thereto, followed by reaction at 37° C. for 1 hour. 10 μl of thereaction product was transformed into DH10B competent cells (Invitrogen,USA) which were then plated on an LB+Ampicillin solid medium andcultured at 37° C. for 20 hours. Colonies grown on the LB+Ampicillinsolid medium were cultured on an LB+Ampicillin liquid medium, followedby DNA purification and sequencing to confirm whether BamHI sitesunderwent point mutations (GGATCC->GGACCC). Using DNA of colonies havingthe point mutation at one restriction site, the other BamHI site wasalso made to have a point mutation according to the same method. Thepoint mutation method used herein was carried out using a Site-DirectedMutagenesis kit (iNtRON).

Primer sequences used in the point mutation of the promoter sequence areas follows.

TABLE 6 SEQ ID Primers NO Forward primer 5′-ACAGCCACGCAGGGTCCTATCT 50for primary GCATG-3′ point mutation Reverse primer5′-CATGCAGATAGGACCCTGCGTG 51 for primary GCTGT-3′ point mutationForward primer 5′-CTCAGTGGGTTAAGGGTCCAGC 52 for secondary ATTGCTGTG-3′point mutation Reverse primer 5′-CACAGCAATGCTGGACCCTTAA 53 for secondaryCCCACTGAG-3′ point mutation

The 3′ arm sequence also has one BamHI site which was thereforepoint-mutated in the same manner as in point mutation of the promoterregion.

Primer sequences used for the point mutation of the 3′ arm sequence areas follows:

TABLE 7 SEQ ID Primers NO Forward primer 5′-GGACAAGAGTGTGGGTCCACTG 54for primary TGGGAAG-3′ point mutation Reverse primer5′-CTTCCCACAGTGGACCCACACT 55 for primary CTTGTCC-3′ point mutation

The porcine beta-casein promoter sequence present in the pGEM-T vectorwas digested with BamHI and XhoI to prepare an 8.3-kb vector. Inaddition, the 3′ arm sequence was digested with XhoI and NotI to preparea 5.0-kb insert (SEQ ID NO: 5). The resulting two restriction fragmentswere ligated to clone a pGEM-T-Pig β casein 5′+3′ vector.

The pBC1 vector was digested with BamHI and NotI to prepare a 10-kbvector, and the pGEM-T-Pig β casein 5′+3′ vector was digested with BamHIand NotI to prepare a 10.3-kb insert. The resulting two restrictionfragments were ligated to construct a pBC1-Pig β casein cloning vector.

The structure of the constructed pBC1-Pig β casein cloning vector isshown in FIG. 2.

In FIG. 2, P_(β-casein) represents a promoter sequence (SEQ ID NO: 2)containing an exon 1 (E1). The exon 1 refers to an exon which is firstarranged in the direction of in the sequence of SEQ ID NO: 1.

In FIG. 2, β-casein 3′ genomic DNA represents a 3′ arm sequence (SEQ IDNO: 5) containing an exon 9 (E9). The exon 9 refers to the 9^(th) exonin the direction of 5′→3′ in the sequence of SEQ ID NO: 1.

Due to having an XhoI restriction site, the gene of a target protein canbe inserted into the vector.

2×3-globin insulator and pBR322 respectively represent an insulator anda vector component derived from the pBC1 vector. Neomycin represents aneomycin-resistant gene which is derived from the pEGFP-N1 vector(Clontech, USA).

The thus-constructed pBC1-Pig β casein vector was deposited with theKorean Collection for Type Cultures (KCTC), the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Daejon, Korea), underAccession Number KCTC 11327BP.

Example 3 Construction of pBC1-Pig β Casein+hEPO-WPRE Vector

Erythropoietin (hEPO) was cloned into a vector having substitution of anampicillin-resistant gene of a pBC1 vector (Invitrogen, USA) with aneomycin-resistant gene {A ‘neo’ gene capable of conferring drugresistance to G418 was obtained from a pEGFP-N1 vector (Clontech, USA)by amplification of a 1.9-kb PCR product (SEQ ID NO: 45) using a forwardprimer 5′-GCGGCCGCGCGCGTCAGGTGGCAC-3′ (SEQ ID NO: 43) and a reverseprimer 5′-CGATCGGACGCTCAGTGGAACGAAAACTC-3′ (SEQ ID NO: 44), and was thencloned into a pGEM T-easy vector. The 1.9-kb neo gene cloned into theT-vector was digested with restriction endonucleases NotI and PvuI toprepare an insert. In addition, an amp gene (ampicillin-resistance gene)region of the pBC1 vector was removed by NotI and PvuI cleavage toprepare a vector. The resulting insert fragment and vector part wereligated to construct a pBC1 vector into which the neo gene(neomycin-resistance gene) was inserted}, followed by replacement of thegoat beta-casein promoter region and the 3′ genomic DNA region presentin the vector with a promoter sequence (SEQ ID NO: 3) and a 3′ armsequence (SEQ ID NO: 5). In addition, expression of hEPO was maximizedby adding to a 3′ side of hEPO, WPRE (woodchuck hepatitis viruspost-transcriptional regulatory element) which is known to augmentprotein expression through stabilization of mRNA.

hEPO and WPRE were each subjected to PCR amplification (PT-200,BIO-RAD). PCR was carried out as follows: one cycle of denaturation at94° C. for 5 minutes; and 35 cycles of denaturation at 94° C. for 30seconds, primer annealing at 56° C. for 30 seconds, and elongation at72° C. for 2.5 minutes for hEPO and 30 seconds for WPRE. Each of theresulting PCR products 2.3 kb (SEQ ID NO: 60) and 0.6 kb (SEQ ID NO: 61)was cloned into a pGEM-T vector (Promega, USA), followed by confirmationof the sequence thereof. The pGEM-T vector harboring hEPO was digestedwith EcoRV and NotI, and the pGEM-T vector harboring WPRE was digestedwith EcoRV and NotI. The resulting two restriction fragments wereligated.

Primer sequences used for PCR amplification of hEPO and WPRE are asfollows.

TABLE 8 SEQ ID Primers NO Forward primer 5′-GGATCCTGTGGTCACCCGGCGCG 56for hEPO C-3′ amplification Reverse primer 5′-GATATCCCATGGGACAGGCTGGC 57for hEPO GCT-3′ amplification Forward primer 5′-GATATCTCTGTTCCTGTTAATCA58 for WPRE ACCTC-3′ amplification Reverse primer5′-GCGGCCGCGAGCCCGAGGCGAAA 59 for WPRE CAG-3′ amplification

The pBC1 vector was digested with BamHI and NotI to remove the goatbeta-casein promoter and the 3′ genomic DAN region, thereby preparing avector. In addition, hEPO+WPRE cloned into the pGEM-T vector wasdigested with BamHI and NotI to prepare a 2.9-kb insert. The resultingvector and insert were ligated to construct pBC1+hEPO-WPRE. For cloningof the promoter and the 3′ arm region into pBC1+hEPO-WPRE, the promotersequence 5.3 kb (SEQ ID NO: 3) and the 3′ arm sequence 5.0 kb (SEQ IDNO: 5) were cloned into a pGEM-T vector (Promega, USA) by means of PCRamplification.

Primer sequences used for PCR amplification of the promoter sequence andthe 3′ arm sequence are as follows.

TABLE 9 SEQ ID Primers NO Forward primer 5′-GGATCCGCTATGCAATCTCATG 62for promoter GAAAG-3′ amplification Reverse primer5′-GGATCCTGACCAGGGTCAACAT 63 for promoter CTACT-3′ amplificationForward primer 5′-GCGGCCGCCTGCACTTCATTCT 64 for 3′ arm CCTGGATAA-3′amplification Reverse primer 5′-GCGGCCGCTTACAGTAAGACCT 65 for 3′ armTCAGGAGCA-3′ amplification

Analogously the procedure of Example 2, point mutations were introducedinto two BamHI sites (GGATCC) present on the porcine beta-caseinpromoter sequence, by a Site-Directed Mutagenesis kit (iNtRON) usingprimers (SEQ ID NO: 50 to SEQ ID NO: 53). The pBC1+hEPO-WPRE vector wasdigested with BamHI, and treated with alkaline phosphatase (CIP) for 30minutes to prepare a vector. In addition, the pGEM-T vector containingthe point-mutated porcine beta-casein 5′ promoter DNA was digested withBamHI to prepare a 5.4-kb insert (SEQ ID NO: 3). The resulting tworestriction fragments were ligated to clone a pBC1-porcine beta-casein5′+EPO-WPRE vector. The pBC1-porcine beta-casein 5′+hEPO-WPRE vector wasdigested with Nod and treated with CIP for 30 minutes to prepare avector. In addition, the pGEM-T vector containing the porcinebeta-casein 3′ arm DNA was digested with Nod to prepare a 5.0-kb insert(SEQ ID NO: 5). The resulting two restriction fragments were ligated toconstruct a pBC1-Pig β casein+hEPO-WPRE vector.

The structure of the constructed pBC1-Pig β casein+hEPO-WPRE vector isshown in FIG. 3.

In FIG. 3, P_(β-casein) represents a porcine beta-casein promotersequence (SEQ ID NO: 3), and β-casein 3′ genomic DNA represents a 3′ armsequence (SEQ ID NO: 5).

hEPO represents a human EPO gene, and WPRE represents a woodchuckhepatitis virus post-transcriptional regulatory element gene.

2×β-globin insulator and pBR322 respectively represent an insulator anda vector component derived from the pBC1 vector. Neomycin represents aneomycin-resistant gene which is derived from the pEGFP-N1 vector(Clontech, USA).

The thus-constructed pBC1-Pig β casein+hEPO-WPRE vector was depositedwith the Korean Collection for Type Cultures (KCTC), the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Daejon, Korea), underAccession Number KCTC 11328BP.

Example 4 Construction of Pig βCasein−hEPO Knock-in Vector Using PorcineBeta-Casein Gene

1) Cloning of pGEM-T-hEPO Vector

For construction of a porcine beta-casein hEPO knock-in vector capableof confirming correct introduction of a gene into a specific site by TKgene selection, two pairs of specific primers (SEQ ID NO: 66 to 68) wereprepared which contain from the beginning of an exon 2 region to aninitiation codon in the porcine beta-casein gene and enablesamplification of a sequence of the hEPO gene from after the initiationcodon. With the above-prepared primer containing the exon 2 region ofporcine beta-casein, primary PCR amplification (PT-200, BIO-RAD) wascarried out from the human genomic DNA. PCR was carried out as follows:one cycle of denaturation at 94° C. for 5 minutes; and 30 cycles ofdenaturation at 94° C. for 30 seconds, primer annealing at 56° C. for 30seconds and elongation at 72° C. for 2.5 minutes. Secondary PCRamplification (PT-200, BIO-RAD) was then carried out using the resultingprimary PCR products as templates. PCR was carried out as follows: onecycle of denaturation at 94° C. for 5 minutes; and 30 cycles ofdenaturation at 94° C. for 30 seconds, primer annealing at 56° C. for 30seconds and elongation at 72° C. for 2.5 minutes.

The PCR-amplification product 2.3-kb hEPO gene (SEQ ID NO: 60)containing the sequence spanning from the porcine beta-casein exon 2region to the initiation codon was cloned into a pGEM-T vector (Promega,USA) to construct a vector (pGEM-T-hEPO).

Primer sequences used for PCR amplification of hEPO are as follows.

TABLE 10 SEQ ID Primers NO First forward 5′-GACTTGATCGCCATGGGGGTGC 66primer for ACGGTGAGTACTC-3′ hEPO amplification Second forward5′-GATATCATTCACAGGACTTGAT 67 primer for CGCCATGGGGG-3′ hEPOamplification Reverse primer 5′-GAATTCATGGGACAGGCTGGCG 68 for hEPOCTGA-3′ amplification

2) Construction of pGEM-T-Pig βcasein 5′ arm and pGEM-T-Pig βcasein 3′arm

In order to clone the promoter sequence (5′ arm) and 3′ arm sequence (3′arm) of the porcine beta-casein gene, primers of SEQ ID NO: 69 to SEQ IDNO: 72 were constructed and PCR amplification was then carried out fromthe porcine genomic DNA. The resulting PCR products 4.9 kb (SEQ ID NO:4) and 5.0 kb (SEQ ID NO: 5) were cloned into a pGEM-T vector to therebyconstruct pGEM-T-Pig βcasein 5′ arm and pGEM-T-Pig βcasein 3′ arm.

TABLE 11 SEQ ID Primers NO Forward primer 5′-GTCGACAGTTGTAGCTGCTGAC 69for promoter CTACAC-3′ amplification Reverse primer5′-GATATCGGGGAAATGAGGGAAA 70 for promoter AAATGTAT-3′ amplificationForward primer 5′-GCGGCCGCCTGCACTTCATTCT 71 for 3′ arm CCTGGATAA-3′amplification Reverse primer 5′-CCGCGGTTACAGTAAGACCTTC 72 for 3′ armAGGAGCA-3′ amplification

3) Construction of Lox A Neo-hEPO Vector

A Lox A neo vector (Gerard Karsenty's, Department of Genetics andDevelopment, College of Physicians and Surgeons, Columbia University,New York, N.Y. 10032) was restricted with EcoRV and EcoRI to prepare avector. In addition, the cloned pGEM-T-hEPO was restricted with EcoRVand EcoRI to prepare a 2.3-kb insert (SEQ ID NO: 60). The resulting tworestriction fragments were ligated to construct a Lox A neo-hEPO vector.

4) Construction of Lox A Neo-hEPO-Poly(A) Vector

In order to insert a poly(A) sequence for stabilization of RNA into a 3′side of the Lox A neo-hEPO vector, the Lox A neo-hEPO vector wasrestricted with EcoRI and treated with alkaline phosphatase for 30minutes to prepare a vector. In addition, the bovine growth hormone(BGH) poly(A) derived from a pcDNA3 vector (Invitrogen, USA) wasrestricted with EcoRI to prepare a 0.3-kb insert. The resulting tworestriction fragments were ligated to construct a Lox A neo-hEPO-poly(A)vector.

5) Construction of Lox A Neo-hEPO-Poly(A)-5′ Arm Vector

In order to insert a Pig β casein 5′ arm into a 5′side of the Lox Aneo-hEPO-poly(A) vector, the Lox A neo-hEPO-poly(A) vector wasrestricted with SalI and EcoRV to prepare a vector. In addition, thecloned pGEM-T-Pig β casein 5′ arm vector was restricted with SalI andEcoRV to prepare a 4.9-kb insert (SEQ ID NO: 4). The resulting tworestriction fragments were ligated to construct a Lox Aneo-hEPO-poly(A)-5′ arm vector.

6) Construction of Lox A Neo-hEPO-Poly(A)-5′ Arm-3′ Arm Vector

In order to insert a Pig β casein 3′ arm into a 3′ side of the Lox Aneo-hEPO-poly(A)-5′ arm vector, the Lox A neo-hEPO-poly(A)-5′ arm vectorwas restricted with NotI and treated with alkaline phosphatase for 30minutes to construct a vector. The cloned pGEM-T-Pig β casein 3′ armvector was restricted with NotI to prepare a 5.0-kb insert (SEQ ID NO:5). The resulting two restriction fragments were ligated to construct aLox A neo-hEPO-poly(A)-5′ arm-3′ arm vector.

7) Construction of Lox A Neo-hEPO-Poly(A)-5′ Arm-3′ Arm-TK Vector

In order to insert a Herpes simplex virus-thymidine kinase (HSV-tk) geneas an apoptotic gene into a 3′ side of the Lox A neo-hEPO-poly(A)-5′arm-3′ arm vector, the Lox A neo-hEPO-poly(A)-5′ arm-3′ arm vector wasrestricted with SacII and treated with alkaline phosphatase for 30minutes to prepare a vector. A pBS-TK vector (Gerard Karsenty's,Department of Genetics and Development, College of Physicians andSurgeons, Columbia University, New York, N.Y. 10032) was restricted withNotI to prepare a 2.3-kb insert (encoding the Herpes simplexvirus-thymidine kinase gene). The resulting two restriction fragmentswere ligated to construct a Lox A neo-hEPO-poly(A)-5′ arm-3′ arm-TKvector (Pig β casein−hEPO knock-in vector).

The structure of the constructed Pig β casein−hEPO knock-in vector isshown in FIG. 4.

In FIG. 4, Pig β casein 5′ arm represents the porcine beta caseinpromoter (SEQ ID NO: 4), and Pig β casein 3′ arm represents the 3′ arm(SEQ ID NO: 5).

hEPO represents a human EPO gene, poly(A) represents a poly(A)signal-encoding gene, Neo cassette represents a neomycin-resistant genewhich serves as a positive selective gene, PGK promoter represents aphosphoglycerate kinase (PGK) promoter, and TK represents a Herpessimplex virus-thymidine kinase (HSV-tk) gene which serves as a negativeselective gene and is derived from the pBS-TK vector.

The thus-constructed Pig β casein−hEPO knock-in vector was depositedwith the Korean Collection for Type Cultures (KCTC), the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Daejon, Korea), underAccession Number KCTC 11329BP.

INDUSTRIAL APPLICABILITY

As apparent from the above description, a porcine beta-casein gene ofthe present invention, a promoter thereof, and an expression vector andtransgenic animal using the same allow for high-concentration secretionof target proteins in milk, which consequently will provide benefits forthe production of useful proteins that are medically andpharmaceutically valuable.

What is claimed is:
 1. An expression vector, comprising one or morenucleotide sequences selected from the group consisting of SEQ ID No. 2,SEQ ID No. 3, and SEQ ID No. 4, wherein the vector further comprises thenucleotide sequence as set forth in SEQ ID No.
 5. 2. The expressionvector of claim 1 wherein the vector further comprises one or moreelements selected from a selective marker, an insulator, and a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE).
 3. Theexpression vector of claim 2, wherein the vector has a cleavage map asshown in FIG.
 2. 4. The expression vector of claim 3, wherein the vectoris pBC1-Pig β-casein.
 5. The expression vector of claim 1 or claim 2,further comprising a target protein-encoding sequence at a 3′ side ofthe promoter sequence.
 6. The expression vector of claim 5, wherein thetarget protein is human erythropoietin (hEPO).
 7. The expression vectorof claim 6, wherein the vector has a cleavage map as shown in FIG.
 3. 8.The expression vector of claim 7, wherein the vector ispBC1-Pig-βcasein+hEPO-WPRE.
 9. The expression vector of claim 1, whereinthe vector is a knock-in vector.
 10. The expression vector of claim 9,wherein the knock-in vector further comprises a selective marker. 11.The expression vector of claim 10, wherein the knock-in vector has acleavage map as shown in FIG.
 4. 12. The expression vector of claim 11,wherein the knock-in vector is Pig-β-casein−hEPO.
 13. A isolatednon-human animal somatic cell, comprising the expression vector of claim1.